The present invention is in the field of dehydrogenases that are related to the retinol dehydrogenase subfamily, recombinant DNA molecules and protein production. The present invention specifically provides novel dehydrogenase polypeptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Dehydrogenases, particularly members of the retinol dehydrogenase subfamilies, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of these subfamily of dehydrogenases. The present invention advances the state of the art by providing a previously unidentified human dehydrogenases that have homology to members of the retinol dehydrogenase subfamilies.
Dehydrogenases
17.beta.-hydroxysteroid dehydrogenase
The enzymes identified as 17.beta.-hydroxysteroid dehydrogenase (HSD) are important in the production of human sex steroids, including androst-5-ene-3.beta.,17.beta.-diol (.DELTA.sup.5-diol), testosterone and estradiol. In humans, several types of 17.beta.-HSD have now been identified and characterized. Each type of 17.beta.-HSD has been found to catalyze specific reactions and to be located in specific tissues. Further information about Types 1, 2 and 3 17.beta.-HSD can be had by reference as follows: Type 1 17.beta.-HSD is described by Luu-The, V. et al., Mol. Endocrinol., 3:1301-1309 (1989) and by Peltoketo, H. et al., FEBSLett, 239:73-77 (1988); Type 2 17.beta.-HSD is described in Wu, L. et al., J. Biol Chem, 268:12964-12969 (1993); Type 3 17.beta.-HSD is described in Geissler, W M, Nature Genetics, 7:34-39 (1994).
Inhibitors of 17.beta.-hydroxysteroid dehydrogenase activity can be used for prophylaxis or treatment of benign prostate hypertrophy (see WO publication 91/100731).
20-alpha-hydroxysteroid dehydroyenase
The enzyme responsible for the ovarian metabolism of progesterone to 20.alpha-hydroxyprogesterone is 20.alpha.-hydroxysteroid dehydrogenase (20.alpha.-HSD). Specifically, 20.alpha.-HSD is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and catalyzes the transfer of hydrogen from NADPH to progesterone.
By metabolizing progesterone to an inactive form, 20.alpha.-HSD plays a central role in inhibiting the maintenance of pregnancy and prevention of implantation [Wiest, Endocrinology 83:1181-184 (1968); Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, i Biochem. J. 0117:193-200 (1970); Rodway and Kuhn, Biochem. J. 152:433-443 (1975)]. Further supporting this role is the fact that it is the increase in ovarian 20.alpha.-HSD activity rather than a decrease in the synthesis of progesterone that contributes to the lower circulating progesterone levels associated with the termination of pregnancy [Kuhn and Briley, Biochem. J. 117:193-201 (1970)]. Indeed, 20.alpha.-HSD gene expression [Albarracin et al. Endocrinology 134:2453-2460 (1994)] and activity remains repressed throughout pregnancy but are induced before parturition [Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, Biochem. J. 117:193-200 (1970]. Also, ovarian 20.alpha.-HSD catalyzes the decline in progesterone levels which occur during normal and induced termination of pregnancy and pseudopregnancy [Hashimoto and Wiest, Endocrinology 84:873-885 (1969); Naito et al., Endocrinology Jpn 33(1):43-50 (February 1986)].
While 20.alpha.-HSD is of much interest as a key enzyme in the termination/prevention of pregnancy, it is possible that the enzyme is also of importance in spontaneous abortions. Specifically, it is possible that a significant number of spontaneous abortions are due to early expression of 20.alpha.-HSD. Therefore, detection of early 20.alpha.-HSD expression would be of interest in those susceptible to early spontaneous abortions. If detection is made early enough, progesterone replacement therapy could be initiated to help maintain the pregnancy.
11.beta.-hydroxysteroid dehydrogenase
Corticosteroids, also referred to as glucocorticoids, are steroid hormones, the most common form of which is cortisol. Modulation of glucocorticoid activity is important in regulating physiological processes in a wide range of tissues and organs. Glucocorticoids act within the gonads to directly suppress testosterone production (Monder et al., 1994). High levels of glucocorticoids may also result in excessive salt and water retention by the kidneys, producing high blood pressure.
Glucocorticoid action is mediated via binding of the molecule to a receptor, such as either a mineralocorticoid receptor (MR) or a glucocorticoid receptor (GR). Krozowski et al. (1983) and Beaumont and Fanestil (1983) showed that MR of adrenalectomised rats have an equal affinity for the mineralocorticoid aldosterone and glucocorticoids, for example corticosterone and cortisol. Confirmatory evidence has been found for human MR (Arriza et al., 1988). In patients suffering from the congenital syndrome of Apparent Mineralocorticoid Excess (AME; Ulick et al., 1979), cortisol levels are elevated and bind to and activate MRs normally occupied by aldosterone, the steroid that regulates salt and water balance in the body. Salt and water are retained in AME patients causing severe hypertension.
The enzyme 11.beta.-hydroxysteroid dehydrogenase (11.beta.HSD) converts glucocorticoids into metabolites that are unable to bind to MRs Edwards et al., 1988; Funder et al., 1988), present in mineralocorticoid target tissues, for example kidney, pancreas, small intestine, colon, as well as the hippocampus, placenta and gonads. For example, in aldosterone target tissues 11.beta.HSD inactivates glucocorticoid molecules, allowing the much lower circulating levels of aldosterone to maintain renal homeostasis. When the 11.beta.HSD enzyme is inactivated, for example in AME patients Ulick et al., 1979) or following administration of glycyrrhetinic acid, a component of licorice, severe hypertension results. Further, placental 11.beta.HSD activity may protect the foetus from high circulating levels of glucocorticoid which may predispose to hypertension in later life Edwards et al., 1993).
Biochemical characterisation of 11.beta.HSD activity indicates the presence of at least two isoenzymes (11.beta.HSD1 and 11.beta.HSD2) with different cofactor requirements and substrate affinities. The 11.beta.HSD1 enzyme is a low affinity enzyme that prefers NADP+ as a cofactor (Agarwal et al., 1989). The 11.beta.HSD2 enzyme is a high affinity enzyme (Km for glucocorticoid=10 nM), requiring NAD+, not NADP+ as the preferred cofactor, belonging to a class of glucocorticoid dehydrogenase enzymes hereinafter referred to as xe2x80x9cNAD+ dependent glucocorticoid dehydrogenasexe2x80x9d enzymes.
Michael et al. (1993) show an inverse correlation between 11.beta.HSD enzyme activity in human granulosa-lutein cells and the success of IVF (in vitro fertilization), and suggest that activity of this enzyme might be related to the success of embryo attachment and implantation following IVF. The measurement of ovarian 11.beta.HSD enzyme activity as a prognostic indicator for the outcome of assisted conception in all species, is the subject of UK Patent Application No 9305984.
3alpha-hydroxysteroid dehydrogenase
Human liver 3alpha-hydroxysteroid plays an important role in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons and in the reduction of ketone-containing drugs (Kume et al., Pharmacogenetics 1999 December; 9(6):763-71). 3alpha-hydroxysteroid is also involved in the metabolism of bile acids (Yamamoto et al., Biol Pharm Bull 1998 November; 21(11):1148-53).
3alpha-hydroxysteroid plays a significant role in 5alpha-dihydrotestosterone metabolism in human liver via 3alpha-hydroxysteroid reduction, followed by subsequent glucuronidation and clearance via the kidney (Pirog et al., J Clin Endocrinol Metab 1999 September; 84(9):3217-21).
Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase
Two major forms of trans-1,2-dihydrobenzene-1,2-diol dehydrogenase exist. One form shows strict specificity for benzene dihydrodiol and NADP+. The other form oxidizes n-butanol, glycerol, sorbitol, and benzene dihydrodiol in the presence of NADP+ or NAD+, and exhibits high reductase activity towards aldehydes, aldoses and diacetyls (Matsuura et al., Biochim Biophys Acta 1987 April 8; 912(2):270-7).
3-oxo-5-beta-steroid 4 dehydrogenase (also referred to as delta 4-3-Ketosteroid 5 beta-reductase)
3-oxo-5-beta-steroid 4 dehydrogenase exhibits activity toward a variety of substrates, including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one (Okuda et al., J Biol Chem 1984 June. 25;259(12):7519-24).
Retinol dehydrogenase
Vitamin A is a pigment essential to vision. Vitamin A comes from the enzymatic conversion of carotenoids, yellow pigments common to carrots and other vegetables, to retinol. Deficiency of vitamin A and insufficient retinol production leads to a variety of maladies in humans and experimental animals. Symptoms of deficiency include vision related disorders such as xerophthalmia and night blindness; dry skin and dry mucous membranes; retarded development and growth; and sterility in male animals.
Cleavage of .beta.-carotene yields two molecules of retinol; oxidation of retinol forms retinal. Retinal and opsin combine to produce rhodopsin, a visual pigment found in nature. The excitation of rhodopsin with visible light triggers a series of photochemical reactions and conformational changes in the molecule which result in the electrical signal to the brain that are the basis of visual transduction (Lehninger et al. (1993) Principles of Biochemistry, Worth Publishers, New York, N.Y.).
Retinol dehydrogenase (RoDH) catalyzes the conversion of retinol to retinal; retinal dehydrogenase converts retinal to retinoate. Retinoate is a retinoid and a hormone which controls numerous biological processes by regulating eukaryotic gene expression. Retinoids, like steroid and thyroid hormones, diffuse directly across the plasma membrane and bind to intracellular receptor proteins. Binding activates the receptors which interact with signaling pathways (Vettermann et al. (1997) Mol. Carcinog. 20: 58-67), and regulate the transcription of specific genes, particularly those mediating vertebrate development (Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York, N.Y.). Retinol is known to be important in epithelial development (Haselbeck et al. (1997) Dev. Dyn. 208: 447-453; and Attar et al. (1997) Mol. Endocrinol. 11: 792-800) and in the development of the central nervous system (Maden et al. (1997) Development 124: 2799-2805). In Maden""s studies on quail embryos, absence of vitamin A, lead to severe deficits including lack of a posterior hindbrain. Conversely, injection of retinol before gastrulation of the embryo prevented positional apoptosis and corrected the CNS defects.
The universal chromophore of visual pigments is 11-cis retinaldehyde which is generated by 11-cis retinol dehydrogenase, a membrane-bound enzyme abundantly expressed in the retinal pigment epithelium of the eye. The gene which encodes 11-cis retinol dehydrogenase may be involved in hereditary eye diseases (Simon et al. (1996) Genomics 36: 424-430).
Chai et al. have identified, cloned, and expressed two isoforms of retinol dehydrogenase, RoDH(I) and (RoDH(II) (1995, J. Biol. Chem. 270: 28408-28412). The deduced amino acid sequence shows that RoDH(I) and RoDH(II) are short-chain dehydrogenases/reductases that share 82% identity. Retinol is the substrate for RoDH(II) which has a higher affinity for NADP than NAD and is stimulated by ethanol and phosphatidyl choline. Although RoDH(II) is not inhibited by the medium-chain alcohol dehydrogenase inhibitor, 4-methylpyrazole, it is inhibited by phenylarsine oxide and carbenoxolone. Chai et al. reported detection of RoDH(I) and RoDH(II) mRNA in rat liver, but RNase protection assays revealed RoDH(I) and RoHD(II) mRNA in kidney, lung, testis, and brain. Based on these data, Chai et al. concluded that RoDH has tissue specific expression.
The retinol signaling pathway plays an important role in human disorders and diseases. Retinoic acid receptors (RARs; -alpha, -beta, and -gamma) are retinoid-activated transcription factors, which mediate effects of retinoids on gene expression. Alterations in receptor expression or function could interfere with the retinoid signaling pathway. Interference with the pathway may enhance cancer development. Vitamin A analogs (retinoids) which interact with RARs, suppress oral and lung carcinogenesis in animal models and prevent the development of tumors in head, neck, and lung cancer patients (Lotan R. 1997 Environ. Heath Perspect. 105 Suppl. 4: 985-988). Lotan reported that RAR beta expression is lost at early stages of carcinogenesis in the aerodigestive tract.
Retinol dehydrogenase may be implicated in embryonic development. The studies of Maden et al. (supra) suggest that retinol may play a significant role in controlling apoptosis during development of the central nervous system. Retinoids are also implicated in epidermal by development. Attar et al. (1997, Mol. Endocrinol. 11: 792-800) showed that disruption of epidermal barrier function results in extremely high incidences of neonatal mortality in pups.
In addition, retinol dehydrogenase activity is linked to hereditary eye diseases (Simon et al. (1996) Genomics 36: 424-430). Autosomal recessive childhood-onset severe retinal dystrophy (arCSRD) is a heterogeneous group of disorders that affect rod and cone photoreceptors simultaneously. Disease genes implicated in arCSRD are expected to encode proteins present in the neuroretina or in the retinal pigment epithelium (RPE). RPE65, a tissue-specific and evolutionarily highly conserved 61 kD protein, is the first disease gene in this group of inherited disorders that is expressed exclusively in RPE, and may play a role in vitamin A metabolism of the retina (Gu et al. (1997) Nat. Genet. 17: 194-197).
Pityriasis rubra pilaris (PRP) is an idiopathic erythematous scaling eruption which can be difficult to distinguish from psoriasis. The expression of RoDH(II) in the retinol signaling pathway may be of pathogenetic importance in the diagnosis of PRP (Magro, C. M. and Crowson, A. N. (1997) J. Cutan. Pathol. 24: 416-424).
The discovery of a new human retinol dehydrogenase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of disorders associated with immune response, cell proliferation, and development.
Substantial chemical and structural homology exists between the protein described herein and 11-cis retinol dehydrogenase (see FIG. 1). 11-cis retinol dehydrogenase are known in the art to be involved in retinal degeneration. For more information relating to the protein of the present invention, see Simon et al., Genomics 1996 September 15;36(3):424-30A, Yamamoto et al., Nat Genet 1999 June; 22(2):188-91H.
Dehydrogenase proteins, particularly members of the retinol dehydrogenase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of dehydrogenase proteins. The present invention advances the state of the art by providing a previously unidentified human dehydrogenase proteins that have homology to members of the retinol dehydrogenase subfamily.
The present invention is based in part on the identification of amino acid sequences of human dehydrogenase polypeptides and proteins that are related to the 11-cis retinol dehydrogenase, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate dehydrogenase activity in cells and tissues that express the dehydrogenase. Experimental data as provided in FIG. 1 indicates expression in the malignant melanoma (metastatic to lymph node), brain (glioblastoma), thyroid, colon tumor (RER+), stomach (poorly differentiated adenocarcinoma with signet ring cell features), primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma and human leukocyte.